Essential Binding Site Research Articles

Dynamics and Metal Ion Binding in the U6 RNA Intramolecular Stem–Loop as Analyzed by NMR

The U6 RNA intramolecular stem-loop (ISL) is a conserved component of the spliceosome, and contains an essential metal ion binding site centered between a protonated adenine, A79, and U80. Correlated with protonation of A79, U80 undergoes a base-flipping conformational change accompanied by significant helical movement. We have investigated the dynamics of the U6 ISL by analyzing the power dependence of 13C NMR relaxation rates in the rotating frame. The data provide evidence that the conformational transition is centered around an exchange lifetime of 84 micros. The U80 nucleotide displays low internal mobility on the picosecond time-scale at pH 7.0 but high internal mobility at pH 6.0, in agreement with the global transition resulting in the base of U80 adopting a looped-out conformation with increased dynamic disorder. A kinetic analysis suggests that the conformational change, rather than adenine protonation, is the rate-limiting step in the pathway of the conformational transition. Two nucleotides, U70 and U80, were found from chemical shift perturbation mapping to interact with the magnesium ion, with apparent K(d) values in the micromolar to millimolar range. These nucleotides also displayed metal ion-induced elevation of R1 rates, which can be explained by a model that assumes dynamic metal ion coordination concomitant with an induced higher shielding anisotropy for the base 13C nuclei. Addition of Mg2+ shifts the conformational equilibrium toward the high-pH (base-stacked) structure, accompanied by a significant drop in the apparent pK(a) of A79.

Requirement for βB1‐crystallin promoter of Xenopus laevis in embryonic lens development and lens regeneration

Regulation of the lens-specific betaB1-crystallin promoter in Xenopus laevis was investigated using transgenic larvae and tadpoles. Comparison of the promoter sequence with that of chicken betaB1-crystallin gene indicates significant sequence similarity over a span of several hundred base pairs starting from the transcriptional start site. Remarkably, PL-1 and PL-2 sequences identified in the chicken promoter as essential binding sites of MAF, Pax6 and Prox1 transcription factors were conserved. Mutations of X (Xenopus) PL-1 and XPL-2 sequences eliminated the promoter activity, indicating a conserved mechanism regulating betaB1-crystallin promoter among vertebrate species. A stepwise deletion of the promoter sequence starting from 2800 bp indicated that the proximal 260 bp directly upstream of the transcription initiation site is sufficient for eliciting lens-specific expression, but the 150 bp promoter sequence is inactive despite it containing the XPL-1 and XPL-2 sequences, suggesting the presence of an additional and essential regulatory sequence located between -150 and -260 bp. Activity of the betaB1-crystallin promoter during lens regeneration from cornea was examined using transgenic tadpoles and found to have the same dependence on promoter regions as in embryonic lens development, indicating that gene regulation is largely shared by the two lens-generating processes.

Molecular and functional analysis of intragenic SMN1 mutations in patients with spinal muscular atrophy

The autosomal recessive spinal muscular atrophy (SMA), a neuromuscular disease and frequent cause of early death in childhood, is caused in 96% of patients by homozygous absence of the survival motor neuron gene (SMN1). The severity of the disease is mainly determined by the copy number of SMN2, a copy gene which predominantly produces exon 7-skipped transcripts and only low amount of full-length transcripts that encode for a protein identical to SMN1. Only about 4% of SMA patients bear one SMN1 copy with an intragenic mutation. A comprehensive molecular genetic analysis of 34 SMA patients who carry one SMN1 gene is presented, including 18 that were previously published. Haplotype analysis with the microsatellite markers Ag1-CA and C212 in these SMA families turned out to be a reliable accessory method in predicting known SMN1 mutations in SMA patients carrying one SMN1 copy. Five novel missense mutations were identified that are localized in: exon 2a c.88G>A (p.D30N) and c.131A>T (p.D44V); exon 3 c.283G>C (p.G95R) and c.332C>G (p.A111G); and exon 6 c.784A>G (p.S262G), respectively. The survival motor neuron (SMN) protein has been shown to be a component of a large complex (termed the SMN complex) that promotes the formation of spliceosomal U small nuclear ribonucleoproteins (snRNPs). Within this complex, SMN forms oligomers and directly interacts via its N-terminus with SMN-interacting protein 1 (SIP1) and via its central Tudor domain with spliceosomal (Sm) proteins. We performed in vitro interaction studies to test whether SMA-causing missense mutations identified in this study interfere with the reported interactions of SMN. Our results show that mutations p.G95R and p.A111G reduce SMN binding to Sm proteins, further confirming the previous finding that the Tudor domain is the essential binding site of SMN to Sm-proteins. However, all mutations, including those in exon 2a, a region shown to be important for the binding of SMN to SIP1, do not disturb the interaction of SMN to SIP1.

Lymnaea EGF and Gigantoxin I, Novel Invertebrate Members of the Epidermal Growth Factor Family

In this review, we compare the sequence and structural relationships of two epidermal growth factor (EGF) family related proteins that have recently been discovered in invertebrate species. The first is L-EGF, a secreted growth factor from the gastropod mollusk Lymnaea stagnalis. The second is a peptide toxin (Gigantoxin I), isolated from the sea anenome Stichodactyla giganteus, which can paralyze crabs. L-EGF and Gigantoxin I share striking sequence similarity with mammalian erbB1 receptor ligands, including most of the essential receptor binding sites. Intriguingly, L-EGF's tertiary structure resembles more the structure of the EGF-like domain of coagulation factors. That is, the secondary and tertiary structure of L-EGF indicates the presence of a double-stranded beta-sheet but also suggests that this protein, in contrast to all other erbB1 ligands, contains a calcium-binding domain. One of the most remarkable features of L-EGF and Gigantoxin I however, is the indication that these protein are synthesized as non-membrane bound secreted peptides. This feature sets L-EGF and Gigantoxin I apart from all other members of the EGF family or EGF-like proteins identified thus far. We discuss sequence similarities and dissimilarities in the light of indications that, despite the more than 600 million years of phylogenetic distance separating both these invertebrates from mammals, Gigantoxin I and L-EGF retain some affinity for the mammalian erbB-family of receptors. Considering that mammalian EGF and its family members are frequently implicated in neoplastic diseases, the increasing number of identified and characterized invertebrate EGF family members may provide valuable leads in the design of erbB receptor antagonists.

Dynamics in the U6 RNA Intramolecular Stem−Loop: A Base Flipping Conformational Change,

The U6 RNA intramolecular stem-loop (ISL) structure is an essential component of the spliceosome and binds a metal ion required for pre-messenger RNA splicing. The metal binding internal loop region of the stem contains a partially protonated C67-(+)A79 base pair (pK(a) = 6.5) and an unpaired U80 nucleotide that is stacked within the helix at pH 7.0. Here, we determine that protonation occurs with an exchange lifetime of approximately 20 micros and report the solution structures of the U6 ISL at pH 5.7. The differences between pH 5.7 and 7.0 structures reveal that the pH change significantly alters the RNA conformation. At lower pH, U80 is flipped out into the major groove. Base flipping involves a purine stacking interaction of flanking nucleotides, inversion of the sugar pucker 5' to the flipped base, and phosphodiester backbone rearrangement. Analysis of residual dipolar couplings as a function of pH indicates that base flipping is not restricted to a local conformational change. Rather, base flipping alters the alignment of the upper and lower helices. The alternative conformations of the U6 ISL reveal striking structural similarities with both the NMR and crystal structures of domain 5 of self-splicing group II introns. These structures suggest that base flipping at an essential metal binding site is a conserved feature of the splicing machinery for both the spliceosome and group II self-splicing introns.

The DNA element controlling expression of the varicella-zoster virus open reading frame 28 and 29 genes consists of two divergent unidirectional promoters which have a common USF site.

The mechanism of the divergent expression of the varicella-zoster virus (VZV) ORF 28 and ORF 29 genes from a common intergenic DNA element, the ORF 28/29 promoter, is of interest based on the observation that both genes are expressed during VZV lytic infection but only the ORF 29 gene is expressed in latently infected neurons. In the work presented here, expression driven by the ORF 28/29 intergenic region was examined. We found that the promoter activity towards the ORF 29 direction is more responsive to activation by the major viral transactivator IE62 than that towards the ORF 28 direction in the context of our experimental system. Analysis of the functional DNA elements involved in IE62 activation of the bidirectional ORF 28/29 regulatory element revealed that in both transfected and VZV-superinfected cells it is a fusion of two unidirectional promoters overlapping an essential USF binding site but with distinct TATA elements. A single TATA element directs expression in the ORF 28 direction, whereas the two TATA elements directing ORF 29 gene expression are alternatively and differentially utilized for transcription initiation. We also identified an Sp1 site localized proximal to the ORF 28 gene which functions as an activator element for expression in both directions. These results indicate that the ORF 28 and ORF 29 genes can be expressed either coordinately or independently and that the observed expression of only the ORF 29 gene during VZV latency may involve neuron-specific cellular factors and/or structural aspects of the latent viral genome.

The Myosin Cardiac Loop Participates Functionally in the Actomyosin Interaction

The motor protein myosin in association with actin transduces chemical free energy in ATP into work in the form of actin translation against an opposing force. Mediating the actomyosin interaction in myosin is an actin binding site distributed among several peptides on the myosin surface including surface loops contributing to affinity and actin regulation of myosin ATPase. A structured surface loop on beta-cardiac myosin, the cardiac or C-loop, was recently demonstrated to affect myosin ATPase and was indirectly implicated in the actomyosin interaction. The C-loop is a conserved feature of all myosin isoforms with crystal structures, suggesting that it is an essential part of the core energy transduction machinery. It is shown here that proteolytic digestion of the C-loop in beta-cardiac myosin eliminates actin-activated myosin ATPase and reduces actomyosin affinity in rigor more than 100-fold. Studies of C-loop function in smooth muscle myosin were also undertaken using site-directed mutagenesis. Mutagenesis of a single charged residue in the C-loop of smooth muscle myosin alters actomyosin affinity and doubles myosin in vitro motility and actin-activated ATPase velocities, thereby involving a charged region of the loop in the actomyosin interaction. It appears likely that the C-loop is an essential electrostatic binding site for actin involved in modulation of actomyosin affinity and regulation of actomyosin ATPase velocity.

Novel inhibition mechanism of Bacillus cereus sphingomyelinase by beryllium fluoride

Phosphate analogs have been known to inhibit competitively various phosphatases and phospholipase C and D. We found for the first time that only beryllium fluoride (BeF x ) among the phosphate analogs studied inhibits Bacillus cereus sphingomyelinase (SMase) activity. The active inhibitory species proved to be not BeF 3 − but BeF 2 by the measurement of SMase activity and of 19F NMR spectroscopy in the presence of a fixed concentration of BeCl 2 and different concentrations of NaF, although both the species have been reported for other kinds of enzymes. The result of kinetic experiment also indicated that the BeF x binds in the vicinity of the essential binding site for the substrate and that the Mg 2+ binding to SMase is essential for the binding of BeF x to the enzyme.

Myocardin is a master regulator of smooth muscle gene expression.

Virtually all smooth muscle genes analyzed to date contain two or more essential binding sites for serum response factor (SRF) in their control regions. Because SRF is expressed in a wide range of cell types, it alone cannot account for smooth muscle-specific gene expression. We show that myocardin, a cardiac muscle- and smooth muscle-specific transcriptional coactivator of SRF, can activate smooth muscle gene expression in a variety of nonmuscle cell types via its association with SRF. Homodimerization of myocardin is required for maximal transcriptional activity and provides a mechanism for cooperative activation of smooth muscle genes by SRF-myocardin complexes bound to different SRF binding sites. These findings identify myocardin as a master regulator of smooth muscle gene expression and explain how SRF conveys smooth muscle specificity to its target genes.

Identification of Novel Domains within Sox-2 and Sox-11 Involved in Autoinhibition of DNA Binding and Partnership Specificity

Sox transcription factors play key regulatory roles throughout development, binding DNA through a consensus (A/T)(A/T)CAA(A/T)G sequence. Although many different Sox proteins bind to this sequence, it has been observed that gene regulatory elements are commonly responsive to only a small subset of the entire family, implying that regulatory mechanisms exist to permit selective DNA binding and/or transactivation by Sox family members. To identify and explore the mechanisms modulating gene activation by Sox proteins further, we compared the function of Sox-2 and Sox-11. This led to the discovery that Sox proteins are regulated differentially at multiple levels, including transactivation, protein partnerships with Pit-Oct-Unc (POU) transcription factors, and DNA binding autoregulation. Specifically, we determined that Sox-11 activates transcription more strongly than Sox-2 and that the transactivation domain of Sox-11 is primarily responsible for this capability. Additionally, we demonstrate that the Sox-11 DNA binding domain is responsible for selective cooperation with the POU factor Brn-2. This requirement cannot be replaced by the DNA binding domain of Sox-2, indicating that the DNA binding domain of Sox proteins is critical for Sox-POU partnerships. Interestingly, we have also determined that a conserved domain of Sox-11 has the novel capability of autoinhibiting its ability to bind DNA in vitro and to activate gene expression in vivo. Our findings suggest that the autoinhibitory domain can repress promiscuous binding of Sox-11 to DNA and plays an important role in regulating the recruitment of Sox-11 to specific genes.

Structural basis for a lethal mutation in U6 RNA.

U6 RNA is essential for nuclear pre-mRNA splicing and has been implicated directly in catalysis of intron removal. The U80G mutation at the essential magnesium binding site of the U6 3' intramolecular stem-loop region (ISL) is lethal in yeast. To further understand the structure and function of the U6 ISL, we have investigated the structural basis for the lethal U80G mutation by NMR and optical spectroscopy. The NMR structure reveals that the U80G mutation causes a structural rearrangement within the ISL resulting in the formation of a new Watson-Crick base pair (C67 x G80), and disrupts a protonated C67 x A79 wobble pair that forms in the wild-type structure. Despite the structural change, the accessibility of the metal binding site is unperturbed, and cadmium titration produces similar phosphorus chemical shift changes for both the U80G mutant and wild-type RNAs. The thermodynamic stability of the U80G mutant is significantly increased (Delta Delta G(fold) = -3.6 +/- 1.9 kcal/mol), consistent with formation of the Watson-Crick pair. Our structural and thermodynamic data, in combination with previous genetic data, suggest that the lethal basis for the U80G mutation is stem-loop hyperstabilization. This hyperstabilization may prevent the U6 ISL melting and rearrangement necessary for association with U4.

Genomics of the marA/soxS/rob regulon of Escherichia coli: identification of directly activated promoters by application of molecular genetics and informatics to microarray data.

Microarray analyses are providing a plethora of data concerning transcriptional responses to specific gene regulators and their inducers but do not distinguish between direct and indirect responses. Here, we identify directly activated promoters of the overlapping marA, soxS and rob regulon(s) of Escherichia coli by applying informatics, genomics and molecular genetics to microarray data obtained by others. Those studies found that overexpression of marA, or the treatment of cells with salicylate to derepress marA, or treatment with paraquat to induce soxS, resulted in elevated transcription of 153 genes. However, only 27 out of the promoters showed increased transcription under at least two of the aforementioned conditions and eight of those were previously known to be directly activated. A computer algorithm was used to identify potential activator binding sites located upstream of the remaining 19 promoters of this subset, and conventional genetic and biochemical approaches were applied to test whether these sites are critical for activation by the homologous MarA, SoxS and Rob transcriptional activators. Only seven out of the 19 promoters were found to be activated when fused to lacZ and tested as single lysogens. All seven contained an essential activator binding site. The remaining promoters were insensitive to stimulation by the inducers suggesting that the great majority of elevated microarray transcripts either were misidentified or resulted from indirect effects requiring sequences outside of the promoter region. We estimate that the total number of directly activated promoters in the regulon is less than 40.

3',5' Cyclic nucleotide phosphodiesterases class III: members, structure, and catalytic mechanism.

3',5' Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of enzymes that were previously divided by their primary structure into two major classes: PDE class I and II. The 3',5' cyclic AMP phosphodiesterase from Escherichia coli encoded by the cpdA gene does not show any homology to either PDE class I or class II enzymes and, therefore, represents a new, third class of PDEs. Previously, information about essential structural elements, substrate and cofactor binding sites, and the mechanism of catalysis was unknown for this enzyme. The present study shows by computational analysis that the enzyme encoded by the E. coli cpdA gene belongs to a family of phosphodiesterases that closely resembles the catalytic machinery known from purple acid phosphatases and several other dimetallophosphoesterases. They share both the conserved sequence motif, D-(X)(n) GD-(X)(n)-GNH[E/D]-(X)(n)-H-(X)(n)-GHXH, which contains the invariant residues forming the active site of purple acid phosphatases, a binuclear Fe(3+)-Me(2+)-containing center, as well as a beta(alpha)beta(alpha)beta motif as a typical secondary structure signature. Furthermore, the known biochemical properties of the bacterial phosphodiesterase encoded by the cpdA gene, such as the requirement of iron ions and a reductant for maintaining its catalytic activity, support this hypothesis developed by computational analysis. In addition, the availability of atomic coordinates for several purple acid phosphatases and related proteins allowed the generation of a three-dimensional model for class III cyclic nucleotide phosphodiesterases.

The role of active site residue arginine 218 in firefly luciferase bioluminescence.

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate firefly luciferin by a sequence of reactions that require Mg-ATP and molecular oxygen. We had previously developed a working model of the luciferase active site based on the X-ray structure of the enzyme without bound substrates. In our model, the side chain guanidinium group of Arg218 appears to be located in close proximity to the substrate's hydroxyl group at the bottom of the luciferin binding pocket. A similar role for Arg337 also has been proposed. We report here the construction, purification, and characterization of mutant luciferases R218A, R218Q, R218K, R337Q, and R337K. Alteration of the Arg218 side chain produced enzymes with 15-20-fold increases in the Km values for luciferin. The contrasting near-normal Km values for luciferin determined with the Arg337 enzymes support our proposal that Arg218 (and not Arg337) is an essential luciferin binding site residue. Bioluminescence emission studies indicated that in the absence of a positively charged group at position 218, red bioluminescence was produced. Based on this result and those of additional fluorescence experiments, we speculate that Arg218 maintains the polarity and rigidity of the emitter binding site necessary for the normal yellow-green emission of P. pyralis luciferase. The findings reported here are interpreted in the context of the firefly luciferase X-ray structures and computational-based models of the active site.

Ets regulation of the erbB2 promoter.

Evaluating the chromatinized erbB2 gene in nuclei from breast cancer cells expressing varying levels of ErbB2 transcripts, we identified a nuclease-sensitive site within a 0.22 kb region of maximum enhancer activity centered over a conserved 28 bp polypurine(GGA)-polypyrimidine(TCC) mirror-repeat and an adjacent essential Ets binding site (EBS). Promoter footprinting with nuclear extracts reveals an intense Ets hypersensitivity site at the EBS whose degree of intensity correlates with the level of cellular ErbB2 expression. In vitro mapping assays show that the supercoiled erbB2 promoter forms an internal triplex structure (Hr-DNA) at the mirror-repeat element. Mutations preventing Hr-DNA formation can enhance erbB2 promoter activity in human breast cancer cells, a result consistent with previous demonstration that Ets-erbB2 promoter complexes cannot form when the mirror-repeat is engaged in triplex binding, and new results suggesting that Ets binding induces severe promoter bending that may restrict local triplex formation. In addition to previously described erbB2-regulating breast cancer Ets factors (PEA3, ESX/Elf-3), Elf-1 is now shown to be another endogenously expressed Ets candidate capable of binding to and upregulating the erbB2 promoter. Given current strategies to transcriptionally inhibit ErbB2 overexpression, including development of novel erbB2 promoter-targeted therapeutics, an EBS-targeted approach is presented using chimeric Ets proteins that strongly repress erbB2 promoter activity.

The Light Chain Binding Domain of Expressed Smooth Muscle Heavy Meromyosin Acts as a Mechanical Lever

Structural data led to the proposal that the molecular motor myosin moves actin by a swinging of the light chain binding domain, or "neck." To test the hypothesis that the neck functions as a mechanical lever, smooth muscle heavy meromyosin (HMM) mutants were expressed with shorter or longer necks by either deleting or adding light chain binding sites. The mutant HMMs were characterized kinetically and mechanically, with emphasis on measurements of unitary displacements and forces in the laser trap assay. Two shorter necked constructs had smaller unitary step sizes and moved actin more slowly than WT HMM in the motility assay. A longer necked construct that contained an additional essential light chain binding site exhibited a 1.4-fold increase in the unitary step size compared with its control. Kinetic changes were also observed with several of the constructs. The mutant lacking a neck produced force at a somewhat reduced level, while the force exerted by the giraffe construct was higher than control. The single molecule displacement and force data support the hypothesis that the neck functions as a rigid lever, with the fulcrum for movement and force located at a point within the motor domain.

CHL1 is a nuclear protein with an essential ATP binding site that exhibits a size-dependent effect on chromosome segregation.

Saccharomyces cerevisiae chl1 mutants have a significant increase in the rate of chromosome missegregation. CHL1 encodes a 99 kDa predicted protein with an ATP binding site consensus, a putative helix-turn-helix DNA binding motif, and homology to helicases. Using site-directed mutagenesis, I show that mutations that are predicted to abolish ATP binding in CHL1 inactivate its function in chromosome segregation. Furthermore, overexpression of these mutations interferes with chromosome transmission of a 125 kb chromosome fragment in a wild-type strain. Polyclonal antibodies against CHL1 show that CHL1 is predominantly in the nuclear fraction of S. CEREVISIAE: CHL1 function is more critical for the segregation of small chromosomes. In chl1Delta1/chl1Delta1 mutants, artificial circular or linear chromosomes <150 kb in size exhibit near random segregation (0.12 per cell division), whereas all chromosomes tested >225 kb were lost at rates (5 x 10(-)(3) per cell division) comparable to that observed for endogenous chromosome III. These results reveal an important role for ATPases/DNA helicases in chromosome segregation. Such enzymes may alter DNA topology to allow loading of proteins involved in maintaining sister chromatid cohesion.

Cooperative action of the catabolite activator protein and AraC in vitro at the araFGH promoter.

Full activation of transcription of the araFGH promoter, p(FGH), requires both the catabolite activator protein (CAP) and AraC protein. At p(FGH), the binding site for CAP is centered at position -41.5, an essential binding site for AraC is centered at position -79.5, and a second, nonessential binding site is centered at position -154.5. In this work, we used the minimal promoter region required for in vivo activation of p(FGH) to examine the roles of CAP and AraC in stimulating formation of open complexes at p(FGH). Migration retardation assays of open complexes showed that RNA polymerase binds exceptionally tightly to the AraC-CAP-p(FGH) complex and that the order of addition of proteins to the initiating complex is important. Similar assays with RNA polymerase containing truncated alpha subunits suggest that AraC interacts with the C-terminal domain of the alpha subunit. Finally, AraC protein also acts to prevent the improper binding of RNA polymerase at a pseudo promoter near the true p(FGH) promoter.

Analysis of measles virus binding sites of the CD46 gene in patients with subacute sclerosing panencephalitis.

Measles virus (MV) binding sites of the CD46 gene were sequenced in patients with subacute sclerosing panencephalitis (SSPE) and in controls. There were 3 novel polymorphisms, including C/T at nucleotide position 38 (C/T38), G/A at position 176 (G/A176), and C/T at position 453 (C/T453), at allele frequencies of.97:.03, .99:.01, and.97:.03, respectively. The G/A176 polymorphism causes an Arg/Gln amino acid change within the essential binding sites of MV, whereas the C/A38 polymorphism causes a Ser/Phe change outside the MV binding sites. The C/T453 polymorphism does not produce an amino acid change. Two of the 40 SSPE patients and 2 of the 32 controls had both C/T38 and C/T453 polymorphisms in heterozygous patterns. One control subject, but no SSPE patients, had the G/A176 polymorphism in a heterozygous pattern. Thus, it is not likely that CD46 gene alteration has a role as a host susceptibility factor in the development of SSPE.

Autoregulation and multiple enhancers control Math1 expression in the developing nervous system.

Development of the vertebrate nervous system requires the actions of transcription factors that establish regional domains of gene expression, which results in the generation of diverse neuronal cell types. MATH1, a transcription factor of the bHLH class, is expressed during development of the nervous system in multiple neuronal domains, including the dorsal neural tube, the EGL of the cerebellum and the hair cells of the vestibular and auditory systems. MATH1 is essential for proper development of the granular layer of the cerebellum and the hair cells of the cochlear and vestibular systems, as shown in mice carrying a targeted disruption of Math1. Previously, we showed that 21 kb of sequence flanking the Math1-coding region is sufficient for Math1 expression in transgenic mice. Here we identify two discrete sequences within the 21 kb region that are conserved between mouse and human, and are sufficient for driving a lacZ reporter gene in these domains of Math1 expression in transgenic mice. The two identified enhancers, while dissimilar in sequence, appear to have redundant activities in the different Math1 expression domains except the spinal neural tube. The regulatory mechanisms for each of the diverse Math1 expression domains are tightly linked, as separable regulatory elements for any given domain of Math1 expression were not found, suggesting that a common regulatory mechanism controls these apparently unrelated domains of expression. In addition, we demonstrate a role for autoregulation in controlling the activity of the Math1 enhancer, through an essential E-box consensus binding site.